This invention relates to a microarray potting instrument (“spotter”).
Microarrays are arrays of very small samples of purified DNA or protein target material arranged as a grid of hundreds or thousands of small spots immobilized onto a solid substrate. The samples in the array are exposed to complementary genetic or protein probe samples derived from cells that have been labeled with fluorescent dyes. DNA probe material selectively binds to target spots only where complementary bonding sites occur, through a process called hybridization. Subsequent quantitative scanning in a fluorescent microarray scanner produces a pixel map of fluorescent intensities. This fluorescent intensity map can be analyzed by special purpose quantitation algorithms which reveal the relative concentrations of the fluorescent probes and hence the level of gene expression, protein concentration, etc. present in the cells from which the probe samples were extracted. The microarray spotter can also be used for other protein reactions.
The microarray substrate is generally made of glass that has been treated chemically to provide for molecular attachment of the spot samples of microarray target material. The microarray substrate is also generally of the same size and shape as a standard microscope slide, i.e., about 25 mm×75 mm×1 mm thick. The array area can extend to within about 1.5 mm of the edges of the substrate. The spots of target material (typically DNA or protein) are approximately round. The spot diameter can vary from about 75 microns to about 500 microns, depending on the dispensing or spotting technique used. There is a general trend toward smaller spots and more compact arrays. The center-to-center spacing between the spots typically falls into the range of 1.5 to 2.5 spot diameters.
The microarray target material is typically stored in 96-well or 384-well plates, with each well in each plate holding a unique target sample in the form of a liquid solution. Some number of these plates are loaded into the microarray spotting instrument. The instrument, using various levels of automation, draws a small amount (0.3–15 microliter) of material into a spot dispenser, and then dispenses spots of much smaller amounts (0.3–5 nanoliter) onto multiple microarray substrates. The spotting instrument may be equipped with multiple (4–64) spot dispensers which allow the spotting of several samples to occur in parallel, thereby increasing instrument throughput.
The general process of spotting, regardless of dispenser type, includes causing the spotting instrument to execute a sequence of spotting cycles, each cycle including dipping a dispenser into the liquid sample in the plate well where some amount is taken up by vacuum aspiration, capillary action or surface tension; moving the dispenser to an appropriate spotting location above a microarray substrate and causing the dispenser to dispense material for one spot; moving the dispenser to a next spotting position and dispensing until all of the spots to be printed with that sample have been dispensed; washing the residual sample from the dispense; drying the dispenser; and repeating the above cycle until all of the desired samples have been dispensed.
Microarray target material can be deposited as spots on substrates by several methods, and spotting instruments utilizing all of these spot dispensers are commercially available. One method works in a manner similar to an ink-jet printer, such that a few microliters of sample are aspirated by vacuum out of a plate well into a hollow tube. The tip of this hollow tube is configured as a nozzle. When the nozzle has been placed at an appropriate location above a microarray substrate, the instrument dispenses the material to form one spot by applying a pulse of pressure to the liquid in the dispensing tube. This dispensing pressure pulse is typically generated by either a combination of a static pressure created upstream by a motorized syringe or other pump or by causing near instantaneous boiling and the momentary opening of a solenoid valve, or by a piezo-electric squeezing pulse applied to a glass capillary tube that forms the nozzle. One or more droplets are ejected, striking the substrate and drying to form a microarray target spot. Ink-jet spotting instruments are typically equipped with 4–8 dispensers.
Another major category of microarray spotting dispenser uses pins. Pin-type microarray spotting instruments are more widely used than ink-jet type instruments and have several advantages in the DNA microarray application. First, pins generally are simpler and quicker to clean between spotting cycles. Cleaning should be done quickly and thoroughly to maintain spotting instrument throughput and to minimize the possibility of cross-contamination between samples. Second, it is fairly straightforward to fit a spotting instrument with dozens of pins—16-pin, 32-pin, 48-pin, and 64-pin printheads are currently commercially available. Inkjet dispensers are generally limited to about 8 per instrument due to their complexity, so a pin-type instrument fitted with many pins can have an advantage in increased microarray throughput. Third, most types of pins are less expensive and require fewer instrument control features than ink-jet microarray spotter dispensers. Ink-jet dispensers are advantageous, however, for spotting viscous or thick microarray target samples, such as proteins, and in applications where the precise volume of liquid dispensed to each spot must be controlled.
Microarray spotting pins have been developed and are commercially available in several distinct forms. The pins can be solid, typically with a shaft diameter of about 1 mm and a tapered point with a small flat on the tip. An area of the flat on the tip (along with the surface properties of the sample liquid and the microarray substrate) determines the size of the spot that the pin forms. Solid pins are simple and robust, but in being dipped into the target material in the well only take up enough material to form one spot. This limitation requires the spotting instrument to dip the pin once for every microarray spot that is to be printed.
Pins that take up sufficient target material to form dozens or hundreds of spots after one dip into the target liquid are also available. One type of pin is formed from a hollow cylindrical tube with an axial slot cut in the tip. This pin draws up sample liquid into the tube and slot by capillary action, and deposits it in much smaller amounts onto the substrate by capillary action upon contact with the microarray substrate. The uptake volume of the pin is sufficient to form dozens of spots by subsequent contact with other microarray substrates in the batch being processed.
Another type of multi-spot dispensing pins is a solid or two-piece pin, with a gap or slot at the tip. This type of pin draws fluid into the gap or slot by capillary action, and deposits a smaller amount onto the substrate by the inertia of the fluid when the pin is rapidly decelerated by lightly tapping it on the substrate. Again, the amount dispensed to form a spot is small compared to the sample uptake volume, so that each dip of the pin into sample liquid takes up enough sample to form dozens of spots.
Yet another type of multi-spot dispensing pin is solid, with a pyramidal taper at a tip that ends in a small, square flat. An even smaller slot is cut across the tip, providing a reservoir for sample liquid. The tip of the pin is then squeezed or bent slightly to bring the two segments of the slotted tip closer together.
In use, these slotted pins are again dipped into sample liquid, so that a fraction of a microliter of the sample is taken up into the slot by capillary action. The specific geometry and material of the pin also causes a very small amount of the bolus of liquid in the slot to wick out onto the two segments of the split pin tip. Then the fluid on the tip of the pin is brought into contact with the microarray substrate where surface energy attracts the fluid on the tip and forms a spot.
These microarray spotting dispensers, whether pins or ink-jet type, are generally mounted in a “printhead” in the spotting instrument. The printhead is typically a solid metal block which holds the dispensers and locates them precisely in three dimensions to facilitate accurate spot placement. The spotting instrument has a processor and positioning system to move the printhead with respect to the source plates of sample liquid, the substrates to be spotted, and the dispenser washing mechanism. Spotting instruments may move the printhead or the substrates and plates in any combination to facilitate the printing motions.
The printhead locates the dispensers on either 4.5 or 9.0 mm center spacing, for example, corresponding to spacing between wells in sample plates. Pin-type printheads are available with between 4 and 64 holes, accommodating between 4 and 64 pins, and ink-jet printheads are available with up to eight dispensers.
The precise location of the spotting dispensers with respect to other features in the spotting instrument should be well-known, with precision of at least a few tens of microns. The dispensers should be precisely centered in each well of the sample plate when taking up sample, as the plate wells are usually of a “v-bottom” type to maximize the ability to take up small volumes. Also, the dispenser washing/drying mechanism generally requires the dispenser to be placed into the centers of holes, the holes being about 1.25 to 2 times the dispenser diameter. Air drawn through these holes dries the dispensers and the dispensers must be well-centered in the holes to provide uniform drying and to prevent collisions with the sides of the holes. The locations of the dispensers with respect to the substrates should be known to a few tens of microns to allow downstream processing of the microarrays (e.g., scanning & quantitation of the scanned images) to be performed with precise knowledge of where the spots are located in the image field.
Spotting instruments are therefore calibrated, i.e., manipulated to cause the printhead and dispensers to move to various sample uptake, washing/drying, and printing positions, where the precision of its location is judged by a technician or some other sensing means external to the instrument, and the instrument control coordinates that correspond to each calibration location are recorded. The recorded calibration locations are utilized by the instrument controller to calibrate all dispenser positioning to be correct for each particular instrument. This calibration process removes the effects of unit-to-unit mechanical or dimensional control tolerances on the dispensers, the printhead, the sample plate holders, the substrate holders, the washer/dryer components, the motion-control mechanism, etc. A skilled technician or engineer is generally required to perform such calibration. The operation can be difficult because the instrument can be damaged easily when performing the motions before calibration is complete and also because judgment of positional accuracy based on magnified visual feedback is often required.
The printhead and dispensers have frequent cleaning and maintenance. Pins need periodic cleaning outside of the instrument. Pins and ink-jet dispensers need to be replaced if they are damaged due to accidental mechanical contact. Unintended spilling, splashing, or spraying of any liquid onto the printhead may also necessitate removing and cleaning of the printhead, especially pin-type printheads which rely on a low-friction slip-fit between the pins and the printhead.
Known mounting mechanism for printheads typically consist of two threaded holes in a nominally flat surface on one side of the printhead and screws that mate with the threaded holes and extend into clearance holes in a printhead mounting bracket on the instrument. When the printhead is removed and replaced, the location and angular tolerances allowed by this type of mounting system are typically 100 to 300 μm and 1 to 3 degrees of angle. The angle of the printhead is important because the dispensers typically protrude between 10 mm and 30 mm from the bottom of the printhead, and angular misalignment of the printhead results in displacements of the dispenser tips.
Another known printhead mount utilizes a locating pin permanently affixed to the printhead, with the axis of the locating pin axis being along the z-axis of the instrument (perpendicular to the surface on which the liquid is dispensed). The pin is held in a bayonet-type latching mechanism in the bracket. The bayonet latching mechanism provides a positive, repeatable location in the z axis by the action of a spring pressing a cross pin into a slot in the bracket mount. The x-axis position and y-axis position are less well controlled, however, due to the clearance between the printhead's bayonet pin and the bracket's socket required for the slip-fit of insertion. θx and θy are likewise not well-controlled due to the radial clearance in the bayonet connection.
The θz position is determined in the bayonet connection by a mechanical stop that is at a small radius (<10 mm) from the axis of the locating pin. Small angular misalignments cause relatively large displacements of the dispensers because the dispensers are located at a radius of about 25–50 mm from the center of the bayonet pin. The precision of this angular stop is limited to a few milliradians, limiting the precision of the x-y location of the dispensing pins to several tens of microns. Because the locating force is provided by a spring rather than the much higher forces delivered by screws, hysteresis of positioning caused by friction is not overcome by this mechanism.